IAJPS 2018, 05 (05), 4153-4157 Muhammad Kamran et al ISSN 2349-7750

CODEN [USA]: IAJPBB ISSN: 2349-7750

INDO AMERICAN JOURNAL OF

PHARMACEUTICAL SCIENCES
http://doi.org/10.5281/zenodo.1252397

Available online at: http://www.iajps.com Research Article

SELECTIVE AND NON-SELECTIVE ACTIVATED AND

INHIBITORY AGENTS EFFECTS ON ADENYLYL CYCLASE
IN THE KIDNEY OF THE RATS
1Muhammad Kamran, 2 Dr. Awais Butt, 3 Shahzaib Nawaz
Medical officer at Trauma Centre Phool Nagar, Kasur, Pakistan
Medical Officer THQ Hospital Sangla Hill
Jinnah Hospital Lahore
Abstract:
Objective: To have in depth knowledge about the effects of nonselective and selective inhibitory and activated
agents on adenylyl cyclase in rat kidney.
Design: A variety of concentrations of pharmacological agents were prepared. They include nebularine, Ap3A,
forskolin, Ap4A and caffeine. Furthermore, effects of these agents were noted in relation to rat kidney adenylyl
activity.
Methods: Tissue of rat kidney was used in the process of preparation of crude extract. Activity of adenylyl cyclase in
connection with crude extract was observed. [2-H3] ATP was used as substrate which ultimately lead to the
formation of cAMP. Pharmacological agents with their prepared concentrations were tested. Prominent activator of
adenylyl cyclase, forskolin, was selected as a compound. Ap4A, caffeine, nebularine, and Ap3A were utilized for
comparison purpose.
Results: Adenylyl cyclase activity was at peak at 100 M forskolin as per concluded results. Nebularine inhibited
activity of enzyme when agent concentration enhanced up to 50 M where the inhibition started to stable. No
considerable effect on the enzyme activity in kidney tissue was observed when caffeine with 10 – 300 M on the of
adenylyl cyclase activity was used. No effect on adenylyl cyclase activity was noted when Ap3A over the
concentration range of 10 – 300 M was used. However, an inhibition effect on the enzyme activity was noted when
Ap4A with the concentration 100 M was used.
Conclusion: Role of cyclic nucleotides in metabolism control and cell-signaling is undeniably significant. It
stimulates inhibitors and activities of cyclase to make some likely physiological impacts. Foreskin being initiator of
adenylyl cyclase, Ap4A and nebularine are established to be latent inhibitors of cyclase, which signifies their
importance in curing schizophrenia, mania, seizure etc.
Key Words: Ap3A, Nucleotides, Caffeine, Forskolin, Ap4A, Nebularine. Adenylyl cyclase
Corresponding author:
Muhammad Kamran, QR code
Medical officer at Trauma Centre Phool Nagar,
Kasur, Pakistan

Please cite this article in press Muhammad Kamran et al., Selective and Non-Selective Activated and Inhibitory
Agents Effects on Adenylyl Cyclase in the Kidney of the Rats, Indo Am. J. P. Sci, 2018; 05(05).

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 IAJPS 2018, 05 (05), 4153-4157 Muhammad Kamran et al
INTRODUCTION:
Cyclic nucleotides have a significant role in
metabolic control and cell-signaling. Inhibitors and
activators related to cyclase are those agents which
have their worth in pharmacology. We are already
quite aware of these compounds. Forskolin is a case
in point in this regard. Activation created by forskolin
is investigated in the study at hand. Other potential
inhibitors and activators in connection with their
effects on kidney adenylyl cyclase were also tested.
The obtained results in terms of the effects of these
compounds i.e. Ap3A, nebularine, Ap4A and
caffeine, were compared to those of forskolin. By
excluding forskolin, all compounds under
investigation were purine derivatives. Nebularine is a
purine nucleoside antibiotic. Purine 9-D-
ribofuranoside is a structure of Nebularine. This
particular inhibitor is entirely rare in naturally
available purine ribosides. Production of compound
as a normal metabolite is done by fungus Lepista
(Clitocybe) Nebularis. It possesses natural potency
just like cytotoxin. Inhibition of saplings growth of
diverse species is carried out by it. It is also
responsible for mitotic abnormality in tips of the
roots. Influenza B virus multiplication is inhibited too
by it [1]. Being enormously poisonous for guinea-
pigs [2] and mice, nebularine creates mitotic
aberrations i.e. breakdown of chromosome. Toxicity
in animal cells is inhibited when ATP, AMP, and
adenosine with concentrations 100-fold that of
nebularine are used. Inhibition of adenosine
deaminase and NAD+ - reliant glucose
dehydrogenase is also carried out by Nebularine [3].
Caffeine is derived mainly from coffee and tea. Being
methylated xanthine, it is observed to block
phosphodiesterase’s [4]. Ap4A and Ap3A are
considered as alarm ones because they carry out
regulation of metabolism of cells, thus indicating the
signals to cells to adjust in new environment. There is
a proof that Ap4A works just like a positive regulator
for DNA duplication [6]. Human platelets contain
massive amount of Ap4A and Ap3A. When platelets
are activated, these two dinucleotides are freed to join
superfluous cellular milieu, where their role in the
regulation of the Vanstone and modulation of platelet
aggregation is significant [7].
MATERIAL AND METHODS:
Sigma Chemical Co. was utilized to get cyclic
nucleotides, biochemicals and column
chromatography materials. Amersham International
plc was used to obtain [23H] adenosine-5-
triphosphate i.e. Ammonium salt. Repeatedly, the
same source (Amersham International plc) was
utilized to get high efficiency ‘Ready Safe’
scintillation cocktail having phenylxylylethane
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ISSN 2349-7750
surfactant. Wister rats (male) utilized to obtain
mammalian tissue, weighing from 250 to 350grams
in average. These were chosen randomly from a rat
colony from a well-known international hospital. The
obtained rats were placed further in animal unit a
well-known international hospital. Air conditioned
(24 degree centigrade) room was provided to them.
12 hours dark/light cycle was maintained too.
Arrangement of feeding consisting of standard food,
were carried out from Grain, Silos and Flour Mills.
Extraction of adenylyl cyclase activity
Collected sampling of tissues were kept in cold place
i.e. ice and were sliced into fragments as per
requirements. Homogenization was completed in
three minutes in ice-cold, 45mM Tris-HCl
(containing pH 07.40) consisting of 8 mM
theophylline, 0.25M sucrose and 6mM 2-mercapto-
ethanol at highest speed i.e. fifteen hundred
revolutions per minute. Nine milliliters of buffer
were utilized for every g of tissue. Centrifugation of
homogentaion was performed at 3000gram for
10mintues at 2 Centigrade. The precipitate was
disposed of while Re-centrifugation of supernatant
was done at 150,000 grams for 40mintues at
temperature two degree centigrade. The concluding
precipitate was re-suspended in two to three
milliliters of 45mM consisting of 8 mM theophylline
and 6mM 2-mercaptoethanol. Division of it was
done into two parts. Out of two parts, one was used to
find out protein while other was utilized for
examination of adenylyl cyclase activity [9]. After
slight adjustments, procedure of Alvarez and Daniels
was utilized [10]. In incubations of assay, 20μl of the
probable inhibitors or activators solution of dissimilar
concentrations replaced 20μl of the Tris-HCl buffer.
RESULTS:
Adenylyl cyclase activity was at peak at 100 M
forskolin as per concluded results. Nebularine
inhibited activity of enzyme when agent
concentration enhanced up to 50 M where the
inhibition started to stable. No considerable effect on
the enzyme activity in kidney tissue was observed
when caffeine with 10 – 300 M on the of adenylyl
cyclase activity was used. No effect on adenylyl
cyclase activity was noted when Ap3A over the
concentration range of 10 – 300 M was used.
However, an inhibition effect on the enzyme activity
was noted when Ap4A with the concentration 100 M
was used.
DISCUSSION:
Present study confirmed that peak activity of
adenylyl cyclase was at 100 M forskolin as in
(Figure. 1). This corresponds to the already known
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 IAJPS 2018, 05 (05), 4153-4157 Muhammad Kamran et al
reports on kidney tissue [11, 12]. In Figure.2,
inhibition of adenylyl cyclase activity by nebularine
is demonstrated. In the earlier stage when nebularine
concentration was enhanced, a quick inhibition
activity was noted up to 50μM nebularine. The
inhibition was noted as 66 percent. Inhibition was
stable from 50 – 300 μM nebularine. Kinetic study
Figure – 1 & 2: The stimulatory effect of forskolin on
the adenylyl cyclase activity (pmol/ min/ mg protein)
of rat kidney. 0.5mM [2-H3] ATP was used as
substrate. Incubation contained 4mM MgSO4, 45 mM
Tris-HCl buffer at pH 7.4. Data points are each the
mean of 12 replicate determination (±SD).
Figure – 3 & 4: Lineweaver-Burk plot of adenylyl
cyclase kinetics in the presence of nebularine. The
enzyme was in 45 mM Tris- HCl buffer (pH 7.4)
containing 10 mM theophylline. Data points are each
of the mean of 12 replicate determination.
This outcome of inhibition of nebularine in relation
to activity of adenylyl cyclase can be because of
GTP- binding regulatory protein effect, e.g.
dissociation of the alpha subunit of the stimulatory
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ISSN 2349-7750
demonstrated that inhibition was non-competitive
when inhibitor concentration was ranging from 0 ,10,
50, 300μM and substrate concentrations ranged from
0.1 to 0.5mM (Figure. 3). Nebularine result on
adenylyl cyclase is akin to the result on xanthine
oxidase i.e. main enzyme of purine catabolism) [13].
GTP- binding regulatory protein can be prevented by
nebularine. In in vivo, nebularine may block hormone
stimulating adenylyl cyclase by stopping receptor.
Inhibitory effects of nebularine in relation to adenylyl
cyclase prove its vitality as a compound in
pharmacological and laboratory procedures. Thus,
possibility of the compound in treating schizophrenia,
mania and seizure etc. cannot be doubted. By
carrying an effective potency of inhibition in case of
adenylyl cyclase, this compound can be taken as
selective inhibitor. No considerable effect on the
enzyme activity in kidney tissue was observed when
caffeine with 10 – 300 M on the of adenylyl cyclase
activity was used(Table-I). However, acquired result
seems in comparison to Sheppard reports [14] and
Jakobs et al. [15]. It is pertinent to note that caffeine
is found to be effective in inhibiting the cAMP-
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induced activation of adenylyl cyclase when it stops induced activation of adenylyl cyclase. After chronic
signal transduction. It does not act directly on caffeine ingestion in mice, forskolin, nonetheless, did
enzymes [16]. Kubota and Oyama [17] observed in not affect stimulation of striatal adenylyl cyclase
the saponin-treated cells, caffeine subdued the cation- [18].
TABLE-I: Caffeine effect on activity of adenylyl cyclase i.e. pmol/min/mg protein, from kidney of rat. 0.5 mM
(2 – H3) ATP and 4mM MgSO4 were combined in incubation mixture. 45 mM Tris-HCl buffer which
contained pH 7.4, was used to extract kidney tissue without having 10mM theophylline. Collected data was
the mean of five rats. Every determination was in triplicate (±SD).
Specific activity
Caffeine (µM) final concentration Change relative to control (%)
(pmol / min / mg protein)
0 (Control) 114 ± 21 0
10 109 ± 18 -5
20 113 ± 19 -1
30 113 ± 15 -1
100 112 ± 16 -2
200 110 ± 13 -4
300 114 ± 17 0
TABLE-II: Ap3A effect on the activity of adenylyl cyclase i.e. pmol/min/mg protein, from kidney of rat. 0.5
mM (2-H3) ATP and 4 mM MgSO4 were combined in incubation mixture. 45 mM Tris-HCl buffer which
contained pH 7.4, was used to extract kidney tissue having 10 mM theophylline. Collected data was the mean
of five rats. Every determination was in triplicate (±SD).
Specific activity (pmol / min /
Ap3A (µM) final concentration Change relative to control (%)
mg protein)
0 (Control) 112 ± 15 0
10 112 ± 11 0
20 113 ± 14 -1
30 103 ± 12 -8
100 104 ± 13 -7
200 107 ± 6 -5
300 107 ± 8 -5
Table – III: Ap4A inhibitory effect on adenylyl cyclase activity i.e. pmol/min/mg protein, from kidney of rat.
0.5 mM (2 – H3) ATP and 4mM MgSO4 were combined in incubation mixture. Kidney buffer, having pH 7.4,
contained 10 mM theophylline. Collected data was the mean of five rats. Every determination was in
triplicate (±SD).
Specific activity
Ap4A (µM) final concentration Change relative to control (%)
(pmol / min / mg protein)
0 (Control) 125 ± 15 0
10 117 ± 7 -6
20 115 ± 13 -8
30 104 ± 6 -17
50 98 ± 4 -22
100 81 ± 5 -35
200 146 ± 17 17
300 133 ± 18 6
1000 123 ± 12 -2

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 IAJPS 2018, 05 (05), 4153-4157 Muhammad Kamran et al
The mixtures of (Ap4A and Ap3A) and (adenylyl
cyclase incubation) were obtained one by one. No
effect on adenylyl cyclase activity was noted when
Ap3A over the concentration range of 10 – 300 M
was used. However, an inhibition effect on the
enzyme activity was noted when Ap4A with the
concentration 100 M was used. These results
indicate that Ap4A and nebularine are latent
inhibitors of cyclase, which signifies their importance
in curing schizophrenia, mania, and seizure.
CONCLUSION:
Role of cyclic nucleotides in metabolism control and
cell-signaling is undeniably significant. It stimulates
inhibitors and activities of cyclase to make some
likely physiological impacts. Foreskin being initiator
of adenylyl cyclase, Ap4A and nebularine are
established to be latent inhibitors of cyclase, which
signifies their importance in curing schizophrenia,
mania, seizure etc.
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